Anti-insulin antibodies arise after administration of exogenous hormone and spontaneously in the autoimmune prodrome of insulin dependent diabetes (IDDM). These autoantibodies are one of the best indicators of beta cell destruction in both human and murine (NOD) diabetes. In this project, we propose to characterize the structure and molecular composition (germline genes, somatic mutation, etc.) of insulin autoantibodies. In our previous studies and preliminary data, we produced anti-insulin mAb using protocols that induce T cell dependent (TD, CFA insulin) and T cell independent (TI, Brucella-insulin) responses in BALB/c mice. Analysis of the IgG1 mAb from TD responses revels some unusual features that include utilization of underrepresented V genes and the presence of tandem prolines in CDR3 of VKs. IgG2 and IgM anti-insulins from TI immunization have characteristics of the preimmune repertoire that include germline encoded V genes, some of which are used by other autoantibodies. Many anti-insulins from both TI and TD repertoires use VK5 related L chains, and these L chains share amino acid sequence motifs with the hormone receptor for insulin. We propose to extend these observations by producing mAb from NOD mice and using reverse transcriptase-polymerase chain reaction (RT/PCR) to study the pathological anti-insulin repertoire. These data will characterize the relationship between the germline repertoire and the repertoires selected by insulin immunization and autoimmune beta cell destruction. Experiments using eukaryotic expression vectors and chain recombination will assess the effect of germline structures and specific motifs (e.g., Pro-Pro) on autoreactivity, epitope specificity, and polyreactivity. These studies will identify structural features of insulin antibodies that may be used to modify adverse immunological reactions and provide new information on genetics and structure of pathological autoantibodies for diagnosis and intervention in autoimmune diabetes.